Luminescence ATP Detection Assay System - PerkinElmer

RE OR S ONLY SE RCH U EA F Luminescence ATP Detection Assay System

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin For best results, see page 15 for product use recommendations. Contents Page 1. Introduction 3 2. Principle 5 3. Advantages of ATPlite TM 7 4. Contents of kit 8 5. Storage 9 6. Safety precautions 10 7. Instrumentation and materials required 10 8. ATPlite assay procedure 11 9. ATP standard 13 10. Recommendations for use 15 11. Ordering information 16 12. References 17

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 2

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 1. Introduction ATPlite TM is an Adenosine TriPhosphate (ATP) monitoring system based on firefly (Photinus pyralis) luciferase. This luminescence assay is the alternative to colorimetric, fluorometric and radioisotopic assays for the quantitative evaluation of proliferation and cytotoxicity of cultured mammalian cells. ATP monitoring can be used to assess the cytocidal, cytostatic and proliferative effects of a wide range of drugs, biological response modifiers and biological compounds 1,2,3,4,5,6. The major advantages of this system are high sensitivity, excellent linearity, simplicity, fast results and the lack of cell harvesting or separation steps. Furthermore, the PerkinElmer ATPlite assay system produces a long lived “glow” type signal with a half-life of greater than five hours, therefore a special luminometer with injectors is not required. The kit is ideal for use with the PerkinElmer luminescence detection instruments in both 96- and 384-well microplates. The simplicity of the ATPlite assay system in a 96-well microplate is illustrated in Figure 1. 3

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 2 300 /1,000 assay: 5 mL 5,000 assay: 25 mL 10,000 assay: 125 mL ATPlite buffer Lyophilized substrate solution Mammalian cell lysis solution 50 μL/well 1 3 50 μL/well CulturPlate (96-well) or ViewPlate (96-well) containing cells (100 μL/well) Seal and mix 4 Measure luminescence Figure 1: ATPlite assay system. 4

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 2. Principle ATP is a marker for cell viability because it is present in all metabolically active cells and the concentration declines very rapidly when the cells undergo necrosis or apoptosis. The ATPlite assay system is based on the production of light caused by the reaction of ATP with added luciferase and d-luciferin. This is illustrated in the following reaction scheme: ATP d-Luciferin O2 LUCIFERASE Mg2 Oxyluciferin AMP PPi CO2 Light The emitted light is proportional to the ATP concentration within certain limits. A limitation associated with common luciferase assay technology is the short half-life of the light emission. This flash-type signal requires luminometers with reagent injectors to measure the quick reaction. ATPlite is a mixture of several substances that extends the signal half-life to over 5 hours. A problem with some ATP assay kits that are currently on the market is that the lysing solutions that release the ATP do not irreversibly inactivate endogenous ATP degrading enzymes (ATPases). Also, some lysing solutions contain chaotropic agents like TCA, which 5

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin have a negative effect on the luciferase activity. The ATPlite kit overcomes these problems by raising the pH of the cell culture medium through the addition of the mammalian cell lysis solution. The lysis solution inactivates the endogenous ATPases. The subsequent addition of the substrate solution (Luciferase/Luciferin) lowers the pH to a suitable level so that the reaction can occur. Figure 2 illustrates the performance of the ATPlite 10 Counts per second (CPS) 10 10 10 10 10 10 8 7 6 5 4 3 2 10 0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 Time (min) Cells / Well 100,000 10,000 1,000 100 10 1 0 Figure 2: Signal stability, dynamic range and linearity of ATPlite with CHO cells cultured and measured in a white 96-well CulturPlate TM from PerkinElmer. 6

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin kit used with a serial dilution of Chinese Hamster Ovary (CHO) cells cultured in dMEM/F12 supplemented with 5% FCS and 1% Pen/Strep (100 µL cell suspension/well). The luminescence was measured on the PerkinElmer TopCount Microplate Scintillation and Luminescence Counter at 22 C. 3. Advantages of ATPlite Long-lived luminescent signal - half-life (t1/2) greater than 5 hours, depending on cell type and medium Rapid - results generated in 15 - 25 minutes Simple and reproducible - no separation steps - only two reagent additions Homogeneous assay - no cell harvesting or centrifugation required Sensitive - down to 5 cells in 100 µL medium (derived from CHO and HL-60 cells in 100 µL medium) Wide linear dynamic range - 10 5 ( as derived from CHO and HL-60 cells) 7

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 4. Contents of kit 6016943 - ATPlite 300 assay kit Each assay kit contains the following components: 1. 2. 3. 4. 5. 1 x 20 mL of mammalian cell lysis solution 1 x 20 mL of substrate buffer solution 3 vials of substrate solution (lyophilized) 1 vial of ATP standard (lyophilized) Instruction booklet 6016941 - ATPLite 1,000 assay kit Each assay kit contains the following components: 1. 2. 3. 4. 5. 1 x 60 mL of mammalian cell lysis solution 1 x 60 mL of substrate buffer solution 10 vials of substrate solution (lyophilized) 2 vials of ATP standard (lyophilized) Instruction booklet 6016947 - ATPLite 5,000 assay kit Each assay kit contains the following components: 1. 2. 3. 4. 5. 8 1 x 270 mL of mammalian cell lysis solution 1 x 270 mL of substrate buffer solution 10 vials of substrate solution (lyophilized) 2 vials of ATP standard (lyophilized) Instruction booklet

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 6016949 - ATPlite 10,000 assay kit Each assay kit contains the following components: 1. 2. 3. 4. 5. 1 x 520 mL of mammalian cell lysis solution 1 x 520 mL of substrate buffer solution 4 bottles of substrate solution (lyophilized) 4 vials of ATP standard (lyophilized) Instruction booklet 5. Storage Upon arrival, store kit at 2 - 8 C. dO NOT FREEZE ! Reconstituted substrate solution can be stored at 2 - 8 C, however the activity declines during storage (approximately 30% lower activity after 1 week). Reconstituted substrate solution can also be stored frozen at - 20 C for longer periods of time. After thawing crystals may appear. These crystals can be dissolved by swirling the vial when it has reached room temperature. 9

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 6. Safety precautions FOR IN VITRO RESEARCH USE ONLY Mammalian cell lysis solution contains 0.1 M of alkaline solution. In case of accidental spillage, wash affected areas thoroughly with water. Good laboratory procedures should be applied for the handling and use of the kit. 7. Instrumentation and materials required 1. detection instrument such as the PerkinElmer TopCount, MicroBeta , LumiCount , VICTOR3TM Multi Label Reader, VICTOR Light, EnVisionTM or EnSpire . 2. Sterile, tissue culture treated, white or black 96- or 384-well microplates such as the PerkinElmer CulturPlate and ViewPlate . 3. ATP-free dispensing materials. 10

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 8. ATPlite assay procedure (for 96-well microplate) 1. Allow the reagents to equilibrate to room temperature. 2. For the 300 and 1,000 assay kit reconstitute one lyophilized substrate solution vial by adding 5 mL of substrate buffer solution. Agitate gently until the solution is homogeneous. For the 5,000 assay kit reconstitute one lyophilized substrate solution vial by adding 25 mL of substrate buffer solution. Agitate gently until the solution is homogeneous. For the 10,000 assay kit reconstitute one lyophilized substrate solution bottle by adding 125 mL of substrate buffer solution. Agitate gently until the solution is homogeneous. 3. Add 50 µL of mammalian cell lysis solution to 100 µL of cell suspension per well of a microplate and shake the plate for five minutes in an orbital shaker at 700 rpm. This lyses the cells and stabilizes the ATP. 4. Add 50 µL substrate solution to the wells and shake the microplate for five minutes in an orbital shaker at 700 rpm. 11

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 5. dark adapt the plate for ten minutes and measure the luminescence. ATPlite general assay procedure microplates is outlined in Figure 3. for Prepare plate with cells, growth factors or cytotoxic agents to a final volume of 100 µL/well Incubate cells according to established procedures Add 50 µL mammalian cell lysis solution and shake five minutes Add 50 µL substrate solution and shake five minutes Dark adapt plate for ten minutes and measure luminescence Figure 3: The ATPlite assay flow chart. 12 96-well

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin Note: Please realize that ATP is everywhere. ATP is the universal energy carrier in nature; both eukaryotes and prokaryotes utilize the molecule for energy storage and transfer. As a result, ATP is abundantly present both in microbial, animal or plant cells and also as free ATP. ATP is fairly heat-stable so mere autoclaving is not always sufficient for complete reduction. Therefore, it is important that direct contact of reagents and hands or fingertips is avoided. Open vials carefully and do not touch the mouth of the bottle. Be careful removing the rubber stoppers from the vials. Use ATP-free pipette tips. Handle microplates carefully and use lids to avoid dust or other contamination. 9. ATP Standard In cases where it is necessary to quantify the ATP released from the cells, perform the following procedure in a 96-well microplate: 1. Reconstitute a vial of lyophilized ATP standard solution with water so that a 10 mM stock solution is obtained. E.g., add 1,170 µL of water if the ATP amount printed on the label is 11.7 µmole or add 960 µL of water if the amount is 9.6 µmole. After addition of the water, allow the ATP to dissolve completely by swirling the vial for one minute. 13

ATPLite booklet contents-NEW 2 ATPLite booklet contents-NEW 2 22-04-15 13:44 Pagin 2. Set up a standard curve in the same microplate that will be used for the experimental samples: a. Take an aliquot of the ATP standard solution and prepare a dilution series in water from a concentration of 1 x 10-5 M down to blank. b. Pipette a series of 100 µL of complete culture medium without cells into the wells of the plate. c. Add to these wells 50 µL of the mammalian cell lysis solution and shake the plate for five minutes in an orbital shaker at 700 rpm. d. Add 10 µL of the ATP dilution series to the wells and shake the plate for five minutes in an orbital shaker at 700 rpm. e. Add 50 µL of the substrate solution and shake for five minutes in an orbital shaker at 700 rpm. f. dark adapt the plate for ten minutes and measure the luminescence. g. Calculate the standard curve. Note: Reconstituted ATP standard is stable for weeks when stored at - 20 C. Diluted ATP solutions are stable for eight hours when stored on ice. 14